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ANALYSIS METHOD point mutations in oncogenes using the technology of mass-spectrometric minisequencing

机译：分析方法使用质谱微测序技术鉴定癌基因中的点突变

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摘要

1. A method for the analysis of somatic point mutations in oncogenes, characterized in that to determine changes in the nucleotide sequence using mass spectrometric technique minisequencing. ! 2. A method according to claim 1, characterized in that to obtain analytically sufficient quantities of the DNA fragments studied genes using polymerase chain reaction carried out in a reaction mixture having the following composition: 66 mM TrisNSl, pH 9.0, 16.6 mM (NH4) 2SO4, 2 mM MgCl2, 100 uM each dNTP, 1 U Taq-polymerase (Promega, USA) and 10 pmol of each oligonucleotide primer. ! 3. A method according to claim 2, characterized in that the oligonucleotide primers were used with the following sequence: RAS_F 5'-GAGAATTCATGACTGAATATAAACTTGT-3 '! RAS_R 5'-ATCGAATTCCTCTATTGTTGGATCATATTC-3 '. ! 4. A method according to claim 1, characterized in that the dephosphorylation of terminal phosphate groups of dNTPs is performed directly in a post-amplification mixture during incubation with 1 unit of shrimp phosphatase activity (Promega, USA) at 37 ° C for 30 min followed by heating inactivation at 85 ° C for 10 min. ! 5. A method according to claim 1, characterized in that the minisequencing reaction is carried out in 10 ul reaction mixture with the following composition: 66 mM Tris-HCl pH 9.0; 16.6 mM (NH4) 2SO4; 2.5 mM MgCl2; containing 0.2 mM dNTPs necessary and / or a ddNTP; 2 U TermiPol DNA Polymerase (Solis Biodyne, Estonia) and 10 pmol of an oligonucleotide probe KRAS + 5'-GTGGTAGTTGGAGCT-3 'or an oligonucleotide probe KRAS- 5'-CAAGGCACTCTTGCCTAC-3'. ! 6. A method according to claim 5, characterized in that the reaction is carried out by minisequencing universal temperature profile: 94 ° C - 20 seconds, 58 ° C - 20 seconds, 72 ° C - 15 seconds for 70 cycles. ! 7. A method according to claim 1, characterized in that for cleaning products in D

机译：1.一种分析癌基因中体细胞点突变的方法，其特征在于使用质谱技术最小测序法确定核苷酸序列的变化。！ 2.根据权利要求1所述的方法，其特征在于，使用在以下组成的反应混合物中进行的聚合酶链式反应，获得分析量足够的所研究的DNA片段：66mM TrisNS1，pH 9.0，16.6mM（NH4） 2SO4、2 mM MgCl2，每个dNTP 100 uM，1 U Taq聚合酶（美国Promega）和每个寡核苷酸引物10 pmol。！ 3.根据权利要求2的方法，其特征在于使用具有以下序列的寡核苷酸引物：RAS_F 5′-GAGAATTCATGACTGAATATAAACTTGT-3′！ RAS_R 5'-ATCGAATTCCTCTATTGTTGGATCATATTC-3'。！ 4.根据权利要求1所述的方法，其特征在于，在1单位虾磷酸酶活性（Promega，USA）在37℃下孵育30分钟的过程中，直接在扩增后混合物中进行dNTPs末端磷酸基团的去磷酸化。然后在85°C加热灭活10分钟。！ 5.根据权利要求1所述的方法，其特征在于，所述微测序反应在具有以下组成的10ul反应混合物中进行：66mM Tris-HCl pH 9.0;和16.6毫米（NH4）2SO4; 2.5毫米氯化镁;包含必需的0.2 mM dNTP和/或ddNTP; 2 U TermiPol DNA聚合酶（爱沙尼亚Solis Biodyne）和10 pmol寡核苷酸探针KRAS + 5'-GTGGTAGTTGGAGCT-3'或寡核苷酸探针KRAS-5'-CAAGGCACTCTTGCCTAC-3'。！ 6.根据权利要求5所述的方法，其特征在于，所述反应是通过对通用温度曲线进行最小排序而进行的：94℃至20秒，58℃至20秒，72℃至15秒持续70个循环。！ 7.根据权利要求1所述的方法，其特征在于，用于清洁D中的产品

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公开/公告号RU2008140067A

专利类型
公开/公告日2010-04-20

原文格式PDF
申请/专利权人 ФЕДЕРАЛЬНОЕ ГОСУДАРСТВЕННОЕ УЧРЕЖДЕНИЕ НАУЧНО-ИССЛЕДОВАТЕЛЬСКИЙ ИНСТИТУТ ФИЗИКО-ХИМИЧЕСКОЙ МЕДИЦИНЫ (ФГУ НИИ ФХМ РОСЗДРАВА) (RU);
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申请/专利号RU20080140067
发明设计人 МОРОШКИНА СВЕТЛАНА ЮРЬЕВНА (RU);ГЕНЕРОЗОВ ЭДУАРД ВИКТОРОВИЧ (RU);КОСТИН ПЕТР АНДРЕЕВИЧ (RU);ЗАХАРЖЕВСКАЯ НАТАЛЬЯ БОРИСОВНА (RU);
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申请日2008-10-10
分类号G01N3/48;C12Q1/68;
国家 RU
入库时间 2022-08-21 18:30:13

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